Novel inhibitors with sulfamethazine backbone: synthesis and biological study of multi-target cholinesterases and α-glucosidase inhibitors

Abstract The underlying cause of many metabolic diseases is abnormal changes in enzyme activity in metabolism. Inhibition of metabolic enzymes such as cholinesterases (ChEs; acetylcholinesterase, AChE and butyrylcholinesterase, BChE) and α-glucosidase (α-GLY) is one of the accepted approaches in the treatment of Alzheimer’s disease (AD) and diabetes mellitus (DM). Here we reported an investigation of a new series of novel ureido-substituted derivatives with sulfamethazine backbone (2a-f) for the inhibition of AChE, BChE, and α-GLY. All the derivatives demonstrated activity in nanomolar levels as AChE, BChE, and α-GLY inhibitors with K I values in the range of 56.07–204.95 nM, 38.05–147.04 nM, and 12.80–79.22 nM, respectively. Among the many strong N-(4,6-dimethylpyrimidin-2-yl)-4-(3-substitutedphenylureido) benzenesulfonamide derivatives (2a-f) detected against ChEs, compound 2c, the 4-fluorophenylureido derivative, demonstrated the most potent inhibition profile towards AChE and BChE. A comprehensive ligand/receptor interaction prediction was performed in silico for the three metabolic enzymes providing molecular docking investigation using Glide XP, MM-GBSA, and ADME-Tox modules. The present research reinforces the rationale behind utilizing inhibitors with sulfamethazine backbone as innovative anticholinergic and antidiabetic agents with a new mechanism of action, submitting propositions for the rational design and synthesis of novel strong inhibitors targeting ChEs and α-GLY. Communicated by Ramaswamy H. Sarma


Introduction
Neurons are unparalleled among cell species in mammalian organisms for their characteristic variety of secretory complexes and their heterogeneity in size and shape. Acetylcholine (ACh) was discovered due to the determination that the inhibitory nerves released a substance inhibiting the heart's chronotropic. Discovered by Loewi as a result of research done with isolated heart preparations in the early 1920s, ACh is the first known neurotransmitter (Pohanka, 2012). It has functions in the peripheral and central nervous system such as attention (Istrefi et al., 2020), motivation (Collins et al., 2016), arousal (Ruivo et al., 2017), memory (Işık et al., 2020c), learning (Provensi et al., 2020), and activate muscles (Benham et al., 1985). People with Alzheimer's disease (AD) have reduced levels of ACh in the brain (Francis, 2005). The only clear case of a transmitter known to be inactivated by the extracellular enzymes is ACh. The enzymes responsible for the break-down of ACh are cholinesterases (ChEs), i.e. acetylcholinesterase (EC 3.1.1.7; acetylcholine hydrolase, AChE) and butyrylcholinesterase (EC 3.1.1.8; acylcholine acylhydrolase, BChE) (Krieger, 2010). On the other hand, AChE is more specific for ACh than BChE (Reale et al., 2018). These enzymes, which possess in various tissues, neurons, and the surrounding extracellular space (Işı k et al., 2017), in many species, including humans, are hydrolyzing the quaternary amine ACh to choline (Ch) and acetic acid within milliseconds (Kessler et al., 2017). However, it remains the capacity of BChE to hydrolyze higher Ch esters, such as butyrylcholine, is a significant physiological function of this enzyme (Chuiko, 2000). ChE inhibitors (ChEIs) increase ACh levels by inhibiting the action of AChE and BChE, which are accountable for the breakdown of Ach . The main use of ChEIs is for the therapy of dementia in patients with AD (Işık, 2019). While numerous ChEIs, in particular, such as donepezil, galantamine, neostigmine, physostigmine, rivastigmine, tacrine are available (Kilic et al., 2020;Mughal et al., 2018;Zaman et al., 2019), tend to reason potentially serious side effects (Mughal et al., 2019), such as vasodilation, slow heart rate, weight loss, constriction of the respiratory tract, constriction of the pupils in the eyes (Schneider, 2000).
It is known that there is a possible relationship between neurodegenerative AD and type 2 diabetes mellitus (DM). Studies in recent years have focused on systems that control synaptic and neuronal functions in the brain. It has been determined that insulin controls synaptic and neuronal functions in the brain. Also, it has been determined that DM patients are more likely to have AD and dementia. In the light of this information, the reason why DM is associated with AD has been expressed as follows; The defect in insulin secretion may be responsible for forming both diseases (Han & Li, 2010). In other words, its deficiency or elevated levels can cause AD and DM. Furthermore, ChE enzymes are known to be associated with diabetes. In a study conducted for this purpose, AChE activity has been higher in streptozotocininduced diabetic rats than in control groups. Moreover, BChE activity was significantly increased in type 1 and type 2 DM compared to control groups (Abbott et al., 1993). These results imply that inhibition of cholinesterase enzymes could be important for treating DM, as in AD patients.
One of the important therapeutic approaches to be considered in DM may be possible with the inhibition of carbohydrate hydrolyzing enzymes such as a-glucosidase (EC 3.2.1.20; a-GLY) (Iftikhar et al., 2019). The a-GLY, which plays an important role in regulating blood glucose levels, is an important enzyme involved in the digestion of carbohydrates (Saleem et al., 2021). a-GLY inhibitors (a-GLYIs) delay the release of D-glucose from carbohydrate-containing diets and the absorption of glucose, thereby lowering plasma glucose levels and suppressing hyperglycemia that can occur at satiety (Ren et al., 2011). As a result of this, a-GLYIs use in DM therapy (Taslimi et al., 2018). Although some a-GLYIs, such as acarbose and voglibose Taha et al., 2021), which are widely used clinically to control blood glucose levels, are effective, they often have side effects such as meteorism and flatulence abdominal distension, and possibly diarrhea (Hollander, 1992;Nakagawa, 2013). In this context, many researchers have turned to the novel drug discovery and development technology and toxicology area to discover alternatives of both ChEs and a-GLY inhibitors (Chen et al., 2013).
Anion transport across cellular membranes is essential for maintaining the normal physiological functions of cells (Poulsen et al., 2010). To date, many small-molecule anion transporters based on urea (Dias et al., 2018), thioureas (Akhtar et al., 2018), sulfonamides (Saha et al., 2016) have been reported (Yu et al., 2019). Moreover, sulfonamide compounds have many biological activities, including anti-Alzheimer's, anticancer, antimicrobial, antiviral, and antidiabetic activities (Figure 1). Thus, sulfonamide compounds derived from urea and its sulfur analogue thiourea have been used continuously to design new bioactive compounds due to their critical pharmacological properties (Akocak et al., 2021;Lolak et al., 2020). In our study, it was synthesized and characterized novel inhibitors with sulfamethazine backbone, which have been important bioactive properties with their chemical structure to discover newly multitarget inhibitors effective on AChE, BChE, and a-GLY. The in silico predictions, namely, prediction of ADME and toxicity profiles, molecular docking, and in vitro inhibition effects, of the synthesized novel ureido-substituted sulfamethazine derivatives (2a-f) were investigated for these enzymes associated with AD and DM.

ChEs and a-GLY kinetic analysis
The inhibition effects of novel ureido-substituted derivatives with sulfamethazine backbone (2a-f) were determined with at least five different inhibitor concentrations on AChE, BChE, and a-GLY. The IC 50 values of the synthesized agents were calculated from Activity (%)-[Ligand] graphs for each derivative according to our previous studies (Akbaba et al., 2013;T€ urkeş, 2019b;T€ urkeş et al., 2015). The inhibition types and K I constants were found by Lineweaver and Burk's curves as described in previous studies (Demir, 2020;T€ urkeş et al., 2014, 2019c. The results were exhibited as mean-± standard error of the mean (95% confidence intervals). Differences between data sets were considered statistically significant when the p-value was less than 0.05.

ADME-Tox study
The ADME-Tox profile screening of the novel ureido-substituted inhibitors with sulfamethazine backbone (2a-f) pertaining to pre-clinical agent discovery stages was performed using the QikProp module  and SwissADME platform (Sever et al., 2020). These ADME-Tox properties include: (i) Molecular weight of the compound; (ii) Computed dipole moment of the compound; (iii) Total solvent-accessible volume in cubic angstroms using a probe with a 1.4 Å Radius; (iv) Octanol/gas partition coefficient; (v) Water/gas partition coefficient; (vi) Octanol/water partition coefficient; (vii) Aqueous solubility; (viii) IC 50 value for the blockage of HERG K þ channels; (ix) Apparent Caco-2 cell permeability in nm/sec; (x) Brain/blood partition coefficient; (xi) Apparent MDCK cell permeability in nm/sec; (xii) Skin permeability; (xiii) Prediction of binding to human serum albumin; (xiv) Human oral absorption; (xv) Van der Waals surface area of polar nitrogen and oxygen atoms; (xvii) Number of violations of Lipinski's rule of five (Lipinski et al., 1997); (xvii) Number of violations of Jorgensen's rule of three (Duffy & Jorgensen, 2000); and (xviii) Pan-assay interference compounds alert.

ChEs and a-GLY inhibition assay
Sulfonamides, which are also called sulfa/sulpha drugs, are synthetic agents containing the sulfonamide chemical group. Sulfa medicines exhibit several activities such as anticonvulsant, anti-bacterial, anti-inflammatory, immunomodulatory, and diuretic effects based on many groups of agents by interfering with cell metabolism. The synthesis of new sulfonamides is carried out to give the compound multi-faceted bioactive properties by adding different groups to known scaffolds. For example, potent some AChE and BChE inhibitors associated with AD that show activity in vivo at low concentrations have been developed by synthesizing several sulfonamide analogs (Kosak et al., 2018). In light of these developments, the researchers have focused on the sulfa drug derivatives synthesized as versatile agents against metabolic diseases in recent years, and many sulfonamide derivatives are improved for the treatment of AD and other central nervous system disorders, various cancer kinds, psychosis, diabetes, and many more complex diseases (Apaydın & T€ or€ ok, 2019; Khanfar et al., 2013). The increase in AChE and BChE activities that hydrolyzes the neurotransmitter ACh causes neurodegenerative diseases such as AD by increasing amyloid protein formation. Strong inhibition of ChEs is an important option to consider when treating the disease to reduce the hydrolysis of this neurotransmitter and accepted as an approach in the treatment of AD (Rao et al., 2007). It may be possible to balance the blood glucose level to a normal level by inhibiting the a-GLY in DM disease, which is known to be associated with AD. It has also been reported that a-GLYIs besides their antidiabetic properties have the potential to treat a variety of diseases, including hepatitis, cancer, and heart conditions (Fischer et al., 1996;McCulloch et al., 1983). In this study, it was set out to investigate the influences of the new inhibitors with sulfamethazine backbone (2a-f) as multi-target cholinesterase and a-glucosidase inhibitors on AChE, BChE, and a-GLY. All the analogues were analyzed for their inhibitory activities versus metabolic enzymes as AChE, BChE, and a-GLY, in comparison with the clinically used medicines tacrine (THA) and acarbose (ACR). The inhibition data (IC 50 and K I values) for all novel ureido-substituted inhibitors with sulfamethazine backbone (2a-f) are summarized in Table 1. AChE was inhibited by all derivatives (2a-f) with a variety of potencies. All novel ureido-substituted inhibitors with sulfamethazine backbone (2a-f) were potent AChE inhibitors with IC 50 s in the range of 72.85-244.38 nM, and K I s ranging between 56.07 ± 9.53 nM and 204.95 ± 11.47 nM. The most active analogues in this series (2a-f) were 4-fluoro substituted derivative 2c and, 3,4-dichloro substituted derivative 2f with K I s of 56.07 ± 9.53 nM, and 64.68 ± 7.08 nM, respectively, compared to standard inhibitor THA (K I of 112.05 ± 24.05 nM). The order of inhibitory activities for the novel ureido-substituted derivatives with sulfamethazine backbone (2a-f) versus AChE decreased in the order of 2c (4-fluoro substituted) > 2f (3,4-dichloro substituted) > 2d (4-chloro substituted) > 2e (4-methyl substituted) > 2a (3-chloro substituted) > 2b (3-methyl substituted).
BChE, one of the other cholinesterases, was inhibited by the novel ureido-substituted inhibitors with sulfamethazine backbone (2a-f) in nanomolar levels with IC 50 s ranging from 49.05 to 203.05 nM, and K I s in the range of 38.05 ± 7.04-147.04 ± 27.06 nM. Moreover, all derivatives (2af) showed more potent inhibitory effects on BChE as compared to THA (K I of 177.15 ± 39.05 nM). Analogue 2c bearing the fluoro moiety at the 4th position was the most potent inhibitor of BChE with a K I of 38.05 ± 7.04 nM, and the second most potent inhibitor was 3,4-dichloro substituted sulfamethazine derivative 2f with a K I of 54.07 ± 9.04 nM. The BChE inhibitory activities for the novel ureido-substituted compounds with sulfamethazine backbone (2a-f) reduced in the order of 2c (4-fluoro substituted) > 2f (3,4-dichloro substituted) > 2a (3-chloro substituted) > 2e (4-methyl substituted) > 2d (4-chloro substituted) >2b (3-methyl substituted). On the other hand, fluoro substitution at the 4th position and 3,4-dichloro substitution made better ChEs inhibitors as compared to other analogues that have been studied in the current work. Moreover, 4-fluoro substituted derivative 2c (K I s for AChE and BChE 56.07 ± 9.53 nM and 38.05 ± 7.04 nM, respectively) was identified as the most potent AChE inhibitor in this series (2a-f).
All the synthesized the novel ureido-substituted inhibitors with sulfamethazine backbone (2a-f) exhibited activity in nanomolar levels as a-GLY inhibitors with the IC 50 and K I values in the range of 9.05-68.76 nM, and 12.80 ± 3.05-79.22 ± 14.66 nM, respectively. Accordingly, these analogues (except compounds 2b and 2e) were determined to be more effective inhibitors, compared to ACR as a standard agent with a K I value of 40.37 ± 3.16 nM. The results revealed that derivatives with chloro moiety at the 3, and 4th position played a crucial role in a-GLY inhibitory activity. In particular, the most active compounds in this series were 3-chloro substituted compound 2a, 4-chloro substituted analogue 2d, and 3,4-dichloro substituted derivative 2f with K I s of 12.80 ± 3.05 nM, 27.65 ± 5.98 nM, and 29.92 ± 3.67 nM, respectively. In this respect, it was found that the inhibitory potency order of the novel ureido-substituted analogues with sulfamethazine backbone (2a-f) was 2a (3-chloro substituted) > 2d (4-chloro substituted) > 2f (3,4-dichloro substituted) > 2c (4-fluoro substituted) > 2e (4-methyl substituted) > 2b (3-methyl substituted).
On the other hand, it showed that the methyl substitution on para-and meta-position dramatically lower the AChE, BChE, and a-GLY activity, as it can be seen in the compounds 2b and 2e. These cases may be useful strategies to develop ChEs and a-GLY inhibitory activity. It was also determined from in silico studies that the presence of these fragments increases the activity. In this direction, many studies have indicated that ureido-substituted sulfamethazine derivatives exhibit significant metabolic enzyme inhibition. In this context, the study by Akocak et al. (2021) reported that a series of six N-carbamimidoyl-4-(3-substitutedphenylureido) benzenesulfonamide derivatives (2a-f) were synthesized, and their inhibition activities investigated for a-GLY, AChE, and BChE by spectrophotometric methods. They found that novel derivatives with sulfaguanidine backbone (2a-f) exhibited effective inhibitory profiles with IC 50 s in the range of 94.38-409.13 nM, and K I s ranging between 103.94 and 491.55 nM against a-GLY, with IC 50 s ranging between 523.05 and 1094.23 nM, K I s in the range of 481.04-913.43 nM versus AChE, and with IC 50 s in the range of 660.40-1058.03 nM, and K I s ranging from 598.47 to 904.73 nM against BChE. Işık et al. (2020c) reported that investigated the effects of some sulfonamide derivatives (S1-S4 and S1i-S4i) on AChE. They found that the synthesized 4-aminobenzenesulfonamides had potential inhibitor properties with different inhibition types for AChE with K I constants in the range of 2.54 ± 0.22-299.60 ± 8.73 lM. Taslimi et al. (2020a) synthesized that the derivatives of amine (1i-11i) and imine sulfonamides (1-11) and investigated the effects of the synthesized derivatives on AChE, a-GLY, and glutathione Stransferase (GST) enzymes. K I values of the series for AChE, a-GLY, and GST were found in the range of 2.26 ± 0. 45-82.46 ± 14.74 lM, 95.73 ± 13.67-1154.65 ± 243.66 lM, and 22.76 ± 1.23-49.29 ± 4.49 lM, respectively. In a study, Gokcen et al. (2016) reported that four groups of sulfonamide derivatives, having isoxazole, pyridine, pyrimidine, thiazole, and thiadiazole groups, synthesized, and their inhibition activities against hCA I and II isoenzymes evaluated. They determined that novel derivatives with sulfamethazine backbone (5c-8c) demonstrated activity in nanomolar levels as hCA I, and hCA II inhibitors with IC 50 values in the range of 9.07-27.72 nM, and 8.77 to 49.51 nM and K I constants in the range of 7.01-27.18 nM, and 5.74-137.96 nM, respectively. In another study, Hamad et al. (2020) reported that a new series of Schiff base derivatives of (E)-4-(benzylideneamino)-N-(4,6-dimethylpyr-imidin-2yl)benzenesulfonamide (3a-3f) were synthesized. Synthesized compounds were evaluated for their Jack Bean urease inhibitory activity. It was determined that all derivatives (3a-3f) showed potent inhibitory activity, ranging between IC 50 s 3.78 ± 3.23 lM and 12.90 ± 2.84 lM, as compared to standard thiourea (IC 50 of 20.03 ± 2.03 lM). Furthermore, to evaluate the drug-likeness of derivatives, ADME prediction was made, and all analogues (3a-3f) were determined to be non-toxic and have passive gastrointestinal absorption. In another study, Tugrak et al. (2020) synthesized the novel compounds with the chemical structure of N-(f4-[N'-(substituted)sulfamoyl]phenylgcarbamothioyl)benzamide (1a-g) and 4-fluoro-N-(f4-[N'-(substituted)sulfamoyl]phenylgcarbamothioyl)benzamide (2a-g) potent and selective hCA I and II isoenzymes inhibitors. The aryl part of compounds 1g and 2g from the derivatives in the series was sulfamethazine. The K I constants of derivative 1g were 59.55 ± 13.07 nM (hCA I) and 12.19 ± 2.24 nM (hCA II), whereas the K I constants of analogue 2g were 55.95 ± 10.72 nM (hCA I) and 47.96 ± 7.91 nM (hCA II). Comparing the K I values of acetazolamide (82.13 ± 4.56 nM for hCA I and 50.27 ± 3.75 nM for hCA II), with compounds 1g and 2g demonstrated promising and selective inhibitory effects against the hCA I and II isoenzymes, the main target proteins.

In silico studies
3.3.1. ADME-Tox study ADME-Tox-related parameters were determined for novel ureido-substituted inhibitors with sulfamethazine backbone (2a-f), and the results are summarized in Table 2. Also, diagrams showing 'drug-likeness' descriptors for 2a and 2c, which are the most active derivatives in this series, are given in Figure 3. None of six ureido-substituted sulfamethazine derivatives (2a-f) were found to have no Lipinski's rule violation, and only two derivatives showed one Jorgensen's rule violation. According to Lipinski's rule, the octanol/water partition coefficient (QPlogPo/w) value should be 5. For these analogues (2a-f) bearing 3-chlorophenyl, m-tolyl, 4-fluorophenyl, 4-chlorophenyl, p-tolyl, and 3,4-dichlorophenyl moiety QPlogPo/w values ranging from 2.10 to 2.72. The predicted number of hydrogen bonds donated (donorHB 5) and the predicted number of hydrogen bonds accepted (accptHB 10) for all the derivatives (2a-f) were in agreement with the drug-likeness requirements of Lipinski's rule of five. Molecular weight (MW) is a crucial factor for binding at the active site. It was found that all compounds (2a-f) have MW between 411.48 and 466.34 (the reference value of MW is < 500 on the report of Lipinski's). The aqueous solubility (QPlogS) of a compound importantly affects its distribution and absorption characteristics. Usually, a high solubility goes along with good absorption. Considering Jorgensen's rule, the QPlogS value should be À5.7. Only compound 2d (QPlogS: À5.79) and compound 2f (QPlogS: À6.07) presented solubility values out of the limits. This is why these two compounds (2d and 2f) displayed one violation of Jorgensen's rule. The predicted apparent Caco-2 cell permeability (QPPCaco values in the range of 156.85-214.39) of the analyzed derivatives (2a-f) indicated excellent results (QPPCaco value should be >22 nm/s) and agreed with Jorgensen's rule of three. To sum up, computed ADME-Tox properties confirmed novel ureido-substituted inhibitors with sulfamethazine backbone (2a-f) as hit-agents displaying suitable druglike properties.

Molecular docking study
To better understand the interaction of the novel ureido-substituted inhibitors with sulfamethazine backbone (2a-f) with 4BDT, 4DBS, and 5NN6, the most potent AChE, BChE, and a-GLY inhibitors 2c (for ChEs) and 2a (for a-GLY) were docked in the binding sites of these enzymes. After that, in the present docking study, the docking patterns of HUW, THA, and MIG were compared with that of  compound 2c for AChE and BChE (K I s: 56.07 ± 9.53 nM and 38.05 ± 7.04 nM, respectively) and compound 2a for a-GLY (K I : 12.80 ± 3.05 nM), the most potent compounds in this series (2a-f). The binding interactions of the inhibitors with AChE, BChE, and a-GLY are displayed in Figure 5.

Conclusion
In conclusion, a series of novel ureido-substituted derivatives with sulfamethazine backbone (2a-f) were synthesized and characterized in detail by spectroscopic and analytic methods. N-(4,6-dimethylpyrimidin-2-yl)-4-(3-substitutedphenylureido) benzenesulfonamide derivatives (2a-f) were first assayed to inhibit AChE, BChE, and a-GLY. The inhibitory activity was more intense versus ChEs compared to standard inhibitors THA and ACR, which, in turn, displayed an inhibition stronger with respect to a-GLY. The effect of derivatives on the enzymes varied according to their molecular structures and positions. Potent novel ureido-substituted derivatives with sulfamethazine backbone (2a-f) were detected towards ChEs with a high interest towards the AChE. A derivative was identified, namely the 4-fluorophenylureido 2c, which displayed a strong inhibitory action versus AChE and BChE. Additionally, an exhaustive in silico ligand/enzyme interaction research was performed with the three metabolic enzymes by Glide XP, MM-GBSA, and ADME-Tox predicts.